Updating the diversity: three novel species of Triblidium (Triblidiaceae, Rhytismatales) in west Yunnan, China

683 MycoKeys

MycoKeys 121: 271-289 (2025)
DOI: 10.3897/mycokeys.121.165642

Research Article

Updating the diversity: three novel species of Triblidium
(Triblidiaceae, Rhytismatales) in west Yunnan, China

Cui-Jin-Yi Li’?*“®, Qi Zhao”®, Prapassorn Damrongkool Eungwanichayapant®, Feng-Ming Yu2®,
Kevin David Hyde?*®, Kandawatte Wedaralalage Thilini Chethana®®, Wei-Wei Liu'®, Dong-Mei Liu'®

1 Institue of Ecology, Chinese Research Academy of Environmental Sciences, Beijing 100012, China
2 State Key Laboratory of Phytochemistry and Natural Medicines, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, China
3 Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100, Thailand
4 School of Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand
Corresponding authors: Dong-Mei Liu (Idmgenetics@163.com); Wei-Wei Liu (liuww@craes. org.cn);
Kandawatte Wedaralalage Thilini Chethana (kandawatte. thi@mfu.ac. th)

OPEN Qrceess

Academic editor: Xin-Cun Wang
Received: 18 July 2025
Accepted: 12 August 2025
Published: 1 September 2025

Citation: Li C-J-Y, Zhao Q,
Eungwanichayapant PD, Yu F-M, Hyde
KD, Thilini Chethana KW, Liu W-W, Liu
D-M (2025) Updating the diversity:
three novel species of Triblidium
(Triblidiaceae, Rhytismatales) in

west Yunnan, China. MycoKeys 121:
271-289. https://doi.org/10.3897/
mycokeys.121.165642

Copyright: © Cui-Jin-Yi Li et al.
This is an open access article distributed under
terms of the Creative Commons Attribution

License (Attribution 4.0 International - CC BY 4.0).

Abstract

During a survey of discomycetes in Yunnan, China, three saprobic species of Triblidium were
discovered on decayed wood and the bark of living oak trees. These species are charac-
terised by black cleistohymenial apothecia with 6-8 teeth-like lobes, which can be either
stipitate or sessile, with greyish-white to orange hymenium, a well-developed covering and
basal stroma, consisting of carbonised to hyaline angular cells or hyaline hyphae, with a sub-
hymenium composed of hyaline angular cells, clavate, J- asci, with an elliptical or rounded
apex and long acicular and ellipsoidal ascospores with multiple septa. Triblidium longispo-
rum, T. stipitatum and T. daliense are described as new species within Triblidium, supported
by both morphological features and phylogenetic analyses of the LSU-ITS-mtSSU dataset.
Detailed descriptions, illustrations and multi-gene analyses fully support each species.

Key words: Phylogeny, Rhytismatales, three new taxa, taxonomy, Triblidiaceae

Introduction

Triblidiaceae Rehm was established within the suborder Triblidieae [Tryblidieae]
under Pezizaceae (Discomycetes) by Rehm (1887-1896). It initially included two
genera: Triblidium Rebent. and Tryblidiopsis P. Karst. (Rehm 1887-1896). Rehm
(1912) included Pseudographis Nyl. and Tryblidiella Sacc in this family. Von H6h-
nel (1918) further broadened the concept of Triblidiaceae, encompassing 11 gen-
era. Nannfeldt (1932) identified Pseudographis as a transitional taxon between
Discomycetes and apothecial lichens. Based on its thick-walled epithecium and
asci, Pseudographis was re-assigned to Lecanorales (Nannfeldt 1932). Subse-
quently, Triblidiaceae was placed within Ostropales Nannf. (Lecanoromycetes)
due to the characteristic reddish-purple, iodine reaction observed in Pseudog-
raphis ascospores (Hawksworth et al. 1983; Sherwood-Pike 1987). Over time,
this reclassification led to only Pseudographis and Triblidium retained within the
family, while Tryblidiopsis was transferred to Rhytismatales (Hawksworth et al.

* These authors have contributed equally to this work and share the first authorship.

2

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

1983; Sherwood-Pike 1987). Eriksson (1992) introduced the genus Huangshania
O.E. Erikss. and placed it within Triblidiaceae under a separate order, Triblidia-
les, to reflect the distant relationship between Triblidium and Graphis Adans., the
type genus of Graphidales. Magnes (1997) extensively revised the family, re-dis-
tributing certain species into 22 genera across seven families. Additionally, he
proposed synonymising Triblidiales with Rhytismatales, based on similarities in
ascomatal development and ascus structures, rejecting any significant relation-
ship between Triblidiaceae and Graphidaceae (Magnes 1997).

Phylogenetic analyses later confirmed the significant divergence of Pseudog-
raphis from Triblidium and Huangshania (Karakehian et al. 2019). As a result,
only Triblidium and Huangshania were retained, while Triblidiales was synony-
mised under Rhytismatales (Karakehian et al. 2019). Based on morphological
similarities, distinguished primarily by ascospore differences and to preserve
the monophyly of Triblidium, Huangshania was synonymised under Triblidium
following a four-locus combined (ITS-LSU-mtSSU-rpb2) phylogenetic analysis.
However, only its type species, Huangshania verrucosa O.E. Erikss., was formal-
ly transferred, as molecular data for the remaining species were unavailable (Lv
et al. 2019). Currently, Triblidiaceae comprises only the type genus, Triblidium
and belongs in Triblidiaceae, Rhytismatales, Leotiomycetes (Hyde et al. 2024).

Triblidium was first established by Rebentisch in 1805 with T. caliciiforme
Rebent. as the type. Later, Rehm (1887-1896, 1912) designated it as the type
genus of the family Triblidiaceae. Triblidium is saprobic and occurs on the bark
of Pinaceae, Ericaceae and Fagaceae, with Magnes (1997) suggesting the pos-
sibility of an endophytic phase. The genus is characterised by circular to rectan-
gular, erumpent apothecia with a rough surface and irregular splits, carbonised
cells or hyphae in the covering stroma, intricate hyphae or angular cells in the
basal stroma, hyaline hyphae within the internal matrix of stroma, intricate hy-
phae or angular cells at the subhymenium, filiform paraphyses, covered with
a thin gelatinous sheath and lack of swelling at the apex, sequentially ripen-
ing asci with J- apex and elliptical, muriform and phragmosporous ascospores
that lack a gelatinous sheath (Eriksson 1992; Magnes 1997; Karakehian et al.
2019; Lv et al. 2019; Guo et al. 2024). A total of 11 species have been described
across America, Asia and Europe (Eriksson 1992; Magnes 1997; Karakehian et
al. 2019; Lv et al. 2019; Guo et al. 2024), with seven species recorded in China
(Eriksson 1992; Karakehian et al. 2019; Lv et al. 2019; Guo et al. 2024).

During a survey of Leotiomycetes diversity in western Yunnan Province, China
(Ekanayaka et al. 2019; Li et al. 2022a, b, 2024a, b; Luo et al. 2024, 2025a, b; Su et
al. 2022, 2023, 2025), five specimens were collected from decayed branches and
the bark of living oak trees. These specimens were identified as three novel spe-
cies of Triblidium. Detailed morphological descriptions, accompanying illustra-
tions and phylogenetic analyses of the new species are presented in this study.

Materials and methods
Sample collection and morphological studies

During the field investigations from 2021 to 2024, all specimens were obtained
from Dali City, Yunnan Province, China. Our collections were obtained from pro-
tected areas and primary forests at altitudes ranging from 2,380 m to 2,600 m.

MycoKeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642 972

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

Sampling methods followed those described by Li et al. (2024b). The dried spec-
imens are deposited at the Herbarium of Cryptogams, Kunming Institute of
Botany, Academia Sinica (HKAS). Facesoffungi numbers were obtained, based
on Jayasiri et al. (2015), while Index Fungorum numbers were obtained following
the guidelines provided by Index Fungorum (2025). Data on the taxa were also
deposited in the Greater Mekong Subregion database (Chaiwan et al. 2021).

Fresh apothecia were photographed in the field with a Canon EOS M100 cam-
era (Canon Co. LTD, Japan). Macro-morphological features of dried apothecia
were captured using a Canon EOS 70D(W) digital camera mounted on a C-PSN
stereomicroscope (Nikon Corporation, Tokyo, Japan). Both fresh and dried apoth-
ecia were manually sliced with razor blades for observation under a charge-cou-
pled device (CCD) SC 2000C attached to a Nikon ECLIPSE Ni-U compound mi-
croscope (Nikon Corporation, Tokyo, Japan). Vertical sections were examined to
study the excipulum and hymenium, while mature apothecia, squashed in water
and 2% potassium hydroxide (KOH) solution, were used to observe asci, aSco-
spores and paraphyses. The blue iodine reaction at the ascus apex was tested
with Melzer's reagent, both in water and 2% KOH solution. All measurements
were made using Tarosoft® Image Framework (IFW) and adjusted in Adobe Pho-
toshop 2020 (Adobe Systems, USA). The Q value represents the ratio of asco-
spore length to width, with ‘n’ indicating the number of measured structures and
Qm denoting the mean Q value. Colour references for the apothecia, hymenium
and excipulum were sourced from https://www.colorhexa.com/.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from multiple mature fruiting bodies using the
Trilief" Plant Genomic DNA Kit (Tsingke Biological Technology Co., Ltd, Bei-
jing, China) following cleaning with sterilised water and a 75% alcohol solution.
The amplification primers for each gene were as follows: ITS1-F and ITS4 for
the nuclear internal transcribed spacers (ITS) (White et al. 1990; Gardes and
Bruns 1993); LROR and LR5 for the D1/D2 domain of the nuclear large subunit
ribosomal RNA (LSU) (Vilgalys and Hester 1990); mrSSU1 and mrSSU3R for
the mitochondrial small subunit (mtSSU rDNA) (Zoller et al. 1999). The 25 ul
total reaction volume consisted of 12.5 ul of 2x Power Taq PCR MasterMix,
7.5 ul of sterile deionised water, 1 ul of each primer (100 uM stock) and 3 ul of
DNA template. PCR amplification was performed using a TC-type gene ampli-
fier (LifeECO, Hangzhou Bori Technology Co., LTD, Hangzhou, China) with the
following conditions: initial denaturation at 94 °C for 5 min, followed by 35 cy-
cles of denaturation at 94 °C for 20 s, annealing at 53 °C for 30 s (for LSU and
ITS) or 56 °C for mtSSU, extension at 72 °C for 45 s and final extension at 72 °C
for 10 min. PCR products were verified by electrophoresis on a 1% agarose gel,
stained with TS-GelRed Ver. 2 (Tsingke Biological Technology Co., Ltd, Beijing,
China) and subsequently sequenced at Tsingke Biological Technology Co., Ltd.

Sequence assembly and alignment

The forward and reverse sequences were assembled using ContigExpress
(Invitrogen, USA) and edited in BioEdit 7.2.5.0 (Hall 1999). The newly-generated
sequences were deposited in GenBank (Table 1) and homologous sequences

MycoKeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642 273

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

Table 1. Detailed information and corresponding GenBank accession numbers for the taxa utilised in the phylogenetic
analyses of this study are provided. ‘” denotes type species, ‘**’ denotes holotypes, newly-generated sequences in bold
font and ‘-’ indicates that sequence data are unavailable.

Gene accession No.

Taxon name Voucher Reference
ITS LSU mtSSU

Fanglania hubeiensis™ HOU 1406 0Q944273 0Q944311 es Guo et al. (2024)
Fanglania parasitica* HOU 1417 0Q944274 0Q944312 0Q944353 Guo et al. (2024)
Neorhytisma panamense HOU 601 GQ253102 GQ253099 0Q944337 Hou et al. (2010)
Neorhytisma panamense* UCH 5284 0Q944277 = 0Q944356 Wang et al. (2023)
Pseudographis elatina GJO-0090016 MK751794 MK751803 MK751717 Karakehian et al. (2019)
Pseudographis elatina PRA-Vondrak25131 0Q718032 = 0Q646404 Vondrak et al. (2023)
Pseudographis pinicola FH-18061 706 MK751795 MK751804 MK751718 Karakehian et al. (2019)
Pseudographis pinicola FH:NB842 MK751796 MK751805 MK751719 Karakehian et al. (2019)
Shuqunia clavata* HOU 1812 PP488619 = = Guo et al. (2024)
Shuqunia longa HOU 457A PP488621 PP488722 PP488816 Guo et al. (2024)
Shuqunia longa™ HOU 368B PP488620 PP488721 PP488815 Guo et al. (2024)
Shuqunia nitens HOU 1845 PP488624 PP488725 PP488819 Guo et al. (2024)
Shuqunia nitens HOU 1758Y PP488623 PP488724 PP488818 Guo et al. (2024)
Shuqunia nitens CNUCC 1758 = PP488666 PP488761 Guo et al. (2024)
Shuqunia nitens* HOU 1758 PP488622 PP488723 PP488817 Guo et al. (2024)
Shuqunia rhododendri CNUCC 1848C.1 PP488554 PP488667 PP488762 Guo et al. (2024)
Shuqunia rhododendri CNUCC 1848C.2 PP488625 PP488726 PP488820 Guo et al. (2024)
Shuqunia rhododendri* HOU 1848D PP488626 PP488727 PP488821 Guo et al. (2024)
Shuqunia yunnanensis HOU 1566 PP488627 PP488728 PP488822 Guo et al. (2024)
Shuqunia yunnanensis HOU 1572 PP488629 PP488730 PP488824 Guo et al. (2024)
Shuqunia yunnanensis HOU 1573 PP488630 = PP488825 Guo et al. (2024)
Shuqunia yunnanensis* HOU 1567 PP488628 PP488729 PP488823 Guo et al. (2024)
Triblidium caliciiformet 7 MK751798 MK751807 MK751721 Karakehian et al. (2019)
Triblidium daliense™ HKAS 128302 PV590251 PV594532 PV593644 This study
Triblidium daliense HKAS 145635 PV590289 PV594530 PV593645 This study
Triblidium hubeiense* HOU 1350A MN541813 MN541811 MN541828 Lv et al. (2019)
Triblidium laojunshanense HOU 2123 PP508362 PP505428 PP505443 Guo et al. (2024)
Triblidium laojunshanense* HOU 1620 PP488635 PP488734 PP488830 Guo et al. (2024)
Triblidium longisporum™ HKAS 145642 PV602119 PV594533 PV593649 This study
Triblidium longisporum HKAS 145640 PV602120 PV594528 PV593648 This study
Triblidium rhododendri HOU 1848B PP488636 PP488735 PP488831 Guo et al. (2024)
Triblidium rhododendri* HOU 326A PP488637 PP488736 PP488832 Guo et al. (2024)
Triblidium rostriforme 1 = MN541820 MN541821 Lv et al. (2019)
Triblidium sichuanense HOU 1964 PP508363 PP505429 PP505444 Guo et al. (2024)
Triblidium sichuanense* HOU 295 PP488639 PP488738 PP488834 Guo et al. (2024)
Triblidium sp. HKAS 128327 PV594526 PV594527 = This study
Triblidium stipitatum™ HKAS 145641 PV594524 PV594531 PV593647 This study
Triblidium stipitatum HKAS 145639 PV594525 PV594529 PV593646 This study
Triblidium verrucosum UME-29336a MK751793 MK751802 MK751716 Karakehian et al. (2019)
Triblidium yunnanense HOU 1611 PP488640 PP488739 PP488835 Guo et al. (2024)
Triblidium yunnanense HOU 1179 MN541814 MN541809 MN541816 Lv et al. (2019)
Tryblidiopsis pinastri* Lantz 412 7 HM140573 = Lantz et al. (2011)
Tryblidiopsis sichuanensis HOU 300 KC312677 KC312679 KC312693 Wang et al. (2014)
Tryblidiopsis sichuanensis* HOU 306 KC312676 KC312683 KC312692 Wang et al. (2014)
Tryblidiopsis sinensis* HOU 814 KC312674 KC312681 KC312694 Wang et al. (2014)

MycoKeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642

274

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

were identified through a BLASTn search against the GenBank database. Phy-
logenetic analyses included related sequences from GenBank, with Fanglania
hubeiensis (Hou 1406) and F. parasiticum (HOU 1417) as the outgroup taxa
(Guo et al. 2024). Sequence alignment for LSU, ITS and mtSSU was performed
using the MAFFT 7 online server (https://mafft.cbrc.jp/alignment/server/)
(Katoh et al. 2019) with default settings and manually refined in BioEdit. The
datasets were trimmed using TrimAl v.1.3, applying the “gt 0.5” option for LSU
and ITS and the ‘gappyout’ option for mtSSU (Capella-Gutiérrez et al. 2009).
Final datasets for each gene were concatenated into a single combined data-
set in the order ‘LSU-ITS-mtSSU’, using SequenceMatrix 1.7.8 (Vaidya et al.
2011) and the file format was converted from “.fasta” to “.nexus” using the
Alignment Transformation Environment online tool (https://www.sing-group.
org/ALTER/). Herbarium abbreviations used in Table 1: CNUCC (Capital Normal
University Culture Collection Center), FH (Harvard University), GJO (Universal-
museum Joanneum), HKAS (Herbarium of Cryptogams, Kunming Institute of
Botany), PRA (Czech Academy of Sciences), UCH (Universidad Autonoma de
Chiriqui) and UME (Umea University).

Phylogenetic analyses

Maximum Likelihood analysis was performed using the IQ-Tree web portal
(http://iqtree.cibiv.univie.ac.at/). The optimal substitution models for each
gene were automatically determined, based on the provided partition file. Clade
support was assessed using a 1,000-replicate SH-aLRT test and the ultrafast
bootstrap (UFB) method (Guindon et al. 2010; Hoang et al. 2018). Bayesian In-
ference (BI) was conducted using MrBayes v.3.1.2. Posterior probabilities (PP)
were estimated via Markov Chain Monte Carlo (MCMC) sampling.

The general time-reversible model with a discrete gamma distribution, cou-
pled with a proportion of an invariant (GTR+I+G) was selected for LSU and mtS-
SU and the unequal transition rates and unequal base frequency model with a
discrete gamma distribution coupled with a proportion of an invariant (HKY+I+G)
was selected for ITS using MrModelTest 2.3 (Nylander et al. 2008). Four simul-
taneous chains were run for 2,000,000 generations, with tree sampling at every
100" generation. An average standard deviation of split frequencies below 0.01
indicated convergence. The first 25% of trees were discarded as burn-in and the
remaining trees were used to estimate PP in the majority-rule consensus tree.
A PP 2 0.90 indicated strong support. The phylogenetic tree was visualised
using FigTree 1.4.0 (Rambaut 2009), illustrated with Adobe Illustrator 2020 and
Photoshop 2020 (https://www.adobe.com/). The combined alignment was de-
posited at TreeBASE (submission ID: 32283).

Results
Phylogenetic analyses

The combined LSU, ITS and mtSSU dataset consists of 23 taxa, including 40
isolates and 2169 aligned nucleotide sites, with the LSU region comprising 856
bp, the ITS region comprising 533 bp and the mtSSU region comprising 780
bp with gaps. The combined alignment comprised 552 parsimony-informative

MycoKeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642 275

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

characters, 179 singleton sites and 1438 constant characters. The ML
and BI analyses yielded similar topologies. The Maximum Likelihood ma-
trix had 710 distinct alignment patterns with 8.48% undetermined charac-
ters or gaps. The best Maximum Likelihood tree, with a final likelihood val-
ue of -11567.119, is shown in Fig. 1. The topology of the phylogenetic tree,
based on the LSU-ITS-mtSSU dataset in this study, closely resembles the
family tree presented by Guo et al. (2024).

Triblidium longisporum and T. stipitatum formed an individual clade with-
in Triblidium close to T. caliciiforme with the Maximum Likelihood bootstrap
support of 83% in the SH-aLRT test, 81% in the UFB method and the Bayesian
posterior probability of 0.91 (Fig. 1). Triblidium daliense is closely related to
T. hubeiense, with the Maximum Likelihood bootstrap support of 79.9% in the
SH-aLRT test, 80% in the UFB method and the Bayesian posterior probability
of 0.90 (Fig. 1). The guidelines of Chethana et al. (2021) and Guo et al. (2024)
were followed in determining whether we had new taxa or records.

Taxonomy

Triblidium daliense C.J.Y. Li, K.W.T. Chethana & Q. Zhao, sp. nov.
Index Fungorum: IF904194

Facesoffungi Number: FoF 18050

Fig. 2

Etymology. The specific epithet refers to Dali City, where the type specimen
was collected.

Holotype. HKAS 128302.

Diagnosis. It is similar to T. sichuanense, but T. daliense has larger asci, wid-
er paraphyses, thicker internal matrix of stroma and the inner layers of the cov-
ering and basal stroma consisting of hyaline hyphae.

Description. Saprobic on the bark of the fallen branches of Fagaceae. Sexual
morph: Apothecia 0.9-1.6 mm wide (x = 1.2 mm,n= 20) when fresh, 1.1-1.6 mm
wide (x = 1.2 mm, n = 20), 0.3-0.5 mm high (x = 0.4 mm, n = 10) when dried,
scattered, superficial, discoid, sessile, erumpent from the bark, initially growing
as acleistohymenial development, the hymenium tightly protected by excipulum
when immature, splitting to expose hymenium by usually irregular 6-8 teeth-
like lobes in the surface in a humid environment, black (#4a4750) surface with
polygonal areolae. Discs flat to slightly raised, circular, orange (#¢c4892d) when
fresh, bright orange (#db9938) when dried. Receptacles rough and black when
fresh, sculptured with polygonal areolae or wrinkled on the surface when dried.
Covering stroma 54-138 um thick, comprised of carbonised textura angularis
cells and the inner layers of hyaline hyphae. Hymenium 200-236 um (x = 216
um, n = 10) thick, hyaline to pale brown. Subhymenium 30-50 um (x = 39 um,
n = 15) thick, comprised of hyaline, textura angularis cells, 3.3-7.9 um (x = 5.3
um, n = 40) in diam. Internal matrix of stroma 55-215 um (x = 123 um, n = 40)
thick, well-developed, comprised of hyaline, textura intricata hyphae, 1.6-2.9
um (x = 2.2 um, n = 40) in diam., non-gelatinous. Basal stroma 60-95 um (x =
74 um, n = 40) thick, well-developed, the outer layers comprised of carbonised,
very dark brown to black, textura angularis cells, 4.3-10.1 um (x = 6.7 um, n
= 40) in diam., the inner layers comprised of highly melanised hyaline hyphae,

Mycokeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642 276

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

0.03 /
a ; re T riblidium hubeiense HOU 1350A H}----- £@
a Se *' | Triblidium yunnanense HOU 1179| oe
ica Triblidium yunnanense HOU 1611 Sa
ee | tg | Triblidium sichuanense HOU 295 H 2 a
Triblidium sichuanense HOU 1964 | =
ee Group ii---+ ee Ue | Triblidium laojunshanense HOU 1620 H i
Pinaceae 97.7/-10.99 oe ----<)
99.9/100/1 NI Triblidium laojunshanense HOU 2123 we
wa Ncaleee 99.9/100/1 Triblidium rhododendri HOU 326A H BA
ee SoGtaeas Triblidium rhododendri HOU 1848B =
Gaknawn ae es Triblidiiim POS YON TRC a As:
substrate | Group III---------- - Triblidium verrucosum UME 29336a }-------- =a
eee soono0n f /“iblidium stipitatum HKAS 145639 TS
Triblidium stipitatum HKAS 145641 T H \
Group IVF. al roo/oor p 2 iblidium longisporum HKAS 145640
Triblidium longisporum HKAS 145642 T H
Group V ---+-----++-- Triblidium calictiforme T |------------------------
Group VI--4---- Triblidium sp. HKAS 128327 t[-----——
Shuqunia nitens CNUCC 1758
96.3/-/-} Shuqunia nitens HOU 1845
99.8/99/1) | Shuqunia nitens HOU 1758Y
877k Shuqunia nitens HOU 1758 H
ae ae Shuqunia yunnanensis HOU 1566
= Shuqunia yunnanensis HOU 1572
Shuqunia yunnanensis HOU 1567 H
| 80.7/95/1 pSAuqunia rhododendri CNUCC 1848C 1
Pee ‘ \oo100/1|t Shuqunia rhododendri CNUCC 1848C 2
[.100/100/1 Shuqunia rhododendri HOU 1848D H
99.9/100/1 Shuqunia longa HOU 457A
Shuqunia longa HOU 368B T H
Shuqunia clavata HOU 1812 H
100/100/1 93.3/97/0.98 7 LVvblidiopsis pinastri Lantz 412 T
99.9/100/1 Tryblidiopsis sinensis HOU 814 H
99.9/100/1 Tryblidiopsis sichuanensis HOU 306 H

Tryblidiopsis sichuanensis HOU 300

100/100/1

Fanglania hubeiensis HOU 1406 T H
Fanglania parasitica HOU 1417H

100/100/1

Neorhytisma panamense HOU 601
Neorhytisma panamense UCH 5284 T

p : o9.o/00/ | L’iblidium daliense HKAS 128302 TH me
TOU De Re aaa alae Triblidium daliense HKAS 145635 ve

Hymenium

JO.

YOO

color

Substrate

Triblidiaceae

Rhytismataceae

Figure 1. Maximum Likelihood tree, based on the LSU, ITS and mtSSU sequences, showing the phylogenetic position of
Triblidium. Bootstrap support values for ML = 80 of SH-aLRT or ML > 95 of UFB and posterior probability for BI = 0.90
are indicated above the nodes and separated by “-/-/-” (SH-aLRT/UFB/PP). The newly-generated isolates of the current
study are highlighted in red, whereas types species within the genera are denoted with ‘T’ and holotypes are denoted as
‘H’ following the strain number. Hymenium colours refer to the illustrations from Lv et al. (2019), Karakehian et al. (2019),

Guo et al. (2024) and this study.

2.9-5.5 um (x = 3.8 um, n = 40) in diam. Paraphyses 270-330 x 2.1-3.3 um
(x = 290 x 2.7 um, n = 50) wide, hyaline with golden oil drops, filiform, apically
irregular-curved and occasionally branched, aseptate. Asci ripening sequential-
ly, 235-292 x 23-28 um (x = 257 x 24 um, n = 25), unitunicate, 8-spored, clav-
ate, apically rounded without an amyloid reaction in Melzer’s reagent, tapering
to a fragile pleurorhynchous subtruncate base, croziers absent. Ascospores
23.4-35.5 x 11.8-16.5 um (x = 26.7 x 13.4 um, n = 55, Q = 1.6-2.2, Qm = 2.0 +
0.1), overlapping uniseriate, ellipsoidal and muriform, hyaline, smooth, slightly
curved, eight transverse septa and one or two longitudinal and oblique septa,
without a gelatinous sheath. Asexual morph: Undetermined.

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277

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

-

Ser eea e255 ee FT ft

Figure 2. Triblidium daliense (HKAS 128302, holotype). A. Fresh apothecia on the substrate; B. Dried apothecia on the
substrate; C. Vertical section of an apothecium; D. Medullary excipulum; E. Ectal excipulum; F. Paraphyses; G. Asci;
H. Ascospores. Scale bars: 500 ym (C); 35 um (D, E); 150 um (F); 100 um (G); 30 um (H).

Material examined. CHINA * Yunnan Province, Dali City, Eryuan County, Ma’an
Mountain, altitude 2,600 m, on the bark of the fallen branches of Fagaceae,
26 July 2021, Cuijinyi Li, LCJY-209 (HKAS 128302, holotype); « ibid., Cuijinyi Li,
LCJY-209-2 (HKAS 145635, isotype).

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Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

Notes. Our collection was placed sister to T. hubeiense, with the Maximum
Likelihood bootstrap support of 79.9% in the SH-aLRT test, 80% in the UFB
method and a Bayesian posterior probability of 0.9 (Fig. 1). Triblidium daliense
can be distinguished from T. hubeiense by its smaller apothecia, thinner cov-
ering stroma (54-138 um vs. 270-300 um), thinner basal stroma (60-95 um
vs. 65-160 um), carbonised angular cells at the outer layer, wider paraphy-
ses (2.1-3.3 um vs. ca. 1 um) with branched tips and larger asci (235-292
x 23-28 um vs. 160-200 x 15-24 um) in contrast to the melanised hyphae
of the latter species at the outer layers (Lv et al. 2019). The most morpholog-
ically similar species to our species is T. sichuanense, which is distinguished
by the presence of angular cells in the inner layers of the covering stroma and
basal stroma, a thinner internal matrix of the stroma (50-80 um vs. 55-215
um), thinner paraphyses (1 um vs. 2.1-3.3 um) and smaller asci (120-220 x
12-20 um vs. 235-292 x 23-28 um) (Guo et al. 2024). Based on the molecular
analyses, the ITS sequence of T. daliense exhibited a 3.3% difference with no
gaps (16/485) to T. yunnanense (isolate: HOU1822B), while the LSU sequence
showed a 2.9% difference with six gaps (26/898) to T. yunnanense (isolate:
HOU875A) and the mtSSU sequence displayed a 0.72% difference with no gaps
(6/830) to T. hubeiense (isolate: HOU1350A).

Triblidium longisporum C.J.Y. Li, K.W.T. Chethana & Q. Zhao, sp. nov.
Index Fungorum: IF904195

Facesoffungi Number: FoF 18051

Fig. 3

Etymology. The specific epithet refers to the long ascospores, ‘longi’ (lat.) =
long, ‘sporum’ (lat.) = spore.

Holotype. HKAS 145642.

Diagnosis. It is similar to T. stipitatum, but T. longisporum has soft orange
hymenium without stipes, simple basal stroma structures and slightly shorter
asci and ascospores.

Description. Saprobic on the bark of the living Fagaceae tree. Sexual
morph: Apothecia 1.3—2.3(—3,1) mm wide (x = 1.8 um, n = 20) when fresh,
1.1-1.9(-2.7) mm wide (x = 1.6 um, n = 20), 0.5-0.8 um high (x = 0.6 um,
n = 10) when dried, scattered, superficial, discoid, sessile, erumpent from
the bark, initially growing as a cleistohymenial development, the hymenium
tightly protected by excipulum when immature, splitting to expose hymenium
by usually 6-8 teeth-like lobes in the surface in a humid environment, black
(#4a4750) surface with polygonal areolae, becoming warty bulges after open-
ing. Discs flat, circular to irregular shape, very soft orange (#dac696) when
fresh, sub-circular to irregular shape, the edges irregularly curl towards the
centre, translucent dark orange (#6d531f) when dried. Receptacles rough and
black when fresh, sculptured with polygonal areolae or wrinkled on the surface
when dried. Lips absent. Covering stroma 80-115(-135) um thick, comprised
of carbonised, textura angularis cells and the inner layers of hyaline, textura
angularis to globulosa cells. Hymenium 330-400 um (x = 360 um, n = 10)
thick, hyaline to pale yellow. Subhymenium 45-85 um (x = 64 um, n = 15)
thick, comprised of hyaline, textura angularis cells, 4.0-9.2 um (x = 6.1 um,

MycoKeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642 279

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

Pas

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on the substrate; F. Vertical section of an apothecium in 2% KOH reagent; G-I. Ectal excipulum in 2% KOH reagent;

B-
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7

Figure 3. Triblidium longisporum (HKAS 145642, holotype). A. Fresh apothecia on the substrate;

reagent; O. Asci in 2% KOH reagent; P. Ascospores (the left in 2% KOH reagent). Scale bars: 1 mm (B); 700 um (C, F);

100 um (N, P); 150 um (0).

M);

70 um (L,

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J

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; 40 um (

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400 um (D

280

MycoKeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

n = 40) in diam. Internal matrix of stroma (110-)140-270 um (x = 195 um,
n = 30) thick, well-developed, comprised of hyaline, textura intricata hyphae,
1.6-3.3 um (x = 2.2 um, n = 40) in diam., non-gelatinous. Basal stroma (40-
)56-128(-154) um (x = 89 um, n = 40) thick, well-developed, the outer lay-
ers comprised of carbonised, gelatinous, black red textura angularis cells,
3.1-6.0(-7.4) um (x = 4.7 um, n = 40) in diam., the inner layers comprised of
hyaline cells, (3.8-)5.1-11.3(-15.7) um (x = 7.2 um, n = 70) in diam., partial
elements orientated at a high angle to receptacle surface, non-gelatinous.
Paraphyses 340-390 x 1.8-2.9 um (x = 360 x 2.3 um, n = 40) wide, hyaline
with some tiny yellow oil drops, filiform, occasionally branched at the tips,
aseptate, apically irregular-shaped and surrounded by a thin, gelatinous
sheath. Asci ripening sequentially, 250-336 x 22-30 um (x = 280 x 26 um,
n = 30), unitunicate, 8-spored, clavate, apically rounded without amyloid re-
action in Melzer’s reagent, tapering to a fragile pleurorhynchous subtruncate
base, croziers absent. Ascospores 160-196 x 5.9-10.5 um (x = 176 x 7.9 um,
n= 40, Q =(16.1)18.8-30.3, Qm = 22.7 + 3.4), overlapping fascicles, long acic-
ular, transverse-septate, hyaline, 28-31-septate when mature, with a single oil
drop in each cell, sharp ends, wide at the top and tapering downwards, thin
and rough-walled with fine verrucae. Asexual morph: Undetermined.

Material examined. CHINA * Yunnan Province, Dali City, Jinguangsi Protec-
tion Zone, altitude 2,380 m, on the living bark of Fagaceae tree, 27 July 2024,
Cuijinyi Li LCJY-1700 (HKAS 145642, holotype); « ibid., Cuijinyi Li LCJY-1691
(HKAS 145640, paratype).

Notes. Our collection was placed sister to T. stipitatum, with the Maximum
Likelihood bootstrap support of 86.5% in the SH-aLRT test, 92% in the UFB
method and a Bayesian posterior probability of 1.0 (Fig. 1). Triblidium longis-
porum can be distinguished from other known species by its exceptionally
long ascospores, with the exception of T. stipitatum. This species differs from
T. stipitatum by its soft orange hymenium, lack of stipes, simple structures at
the covering and basal stroma, smaller asci (250-336 um vs. 272-355 um)
and shorter ascospores (160-196 um vs. 187-226 um). Based on the molec-
ular analyses, the ITS sequence of T. longisporum exhibited a 11.4% difference
with 34 gaps (60/523) to T. stipitatum (isolate: HKAS 145641), while the LSU
sequence showed a 5.7% difference with one gap (48/838) to T. stipitatum (iso-
late: HKAS 145641) and the mtSSU sequence displayed a 3.7% difference with
10 gaps (31/831) to T. stipitatum (isolate: HKAS 145641).

Triblidium stipitatum C.J.Y. Li, K.W.T. Chethana & Q. Zhao, sp. nov.
Index Fungorum: IF904196

Facesoffungi Number: FoF 18052

Fig. 4

Etymology. The specific epithet refers to the presence of stipes, ‘stipitatum’
(lat.) = stipitate.

Holotype. HKAS 145641.

Diagnosis. It is similar to T. longisporum, but differs by having a stipitate,
greyish-white disc, a basal stroma, consisting of three parts and slightly longer
asci and ascospores.

MycoKeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642 281

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

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Figure 4. Triblidium stipitatum (HKAS 145641, holotype). A, B. Fresh apothecia on the substrate; C, D. Dried apothecia on

the substrate; E. Vertical section of an apothecium in 2% KOH reagent; F—H. Ectal excipulum; G-H. Ectal excipulum in 2%
KOH reagent; I. Medullary excipulum in 2% KOH reagent; J, K. Subhymenium in 2% KOH reagent; L. Paraphyses; M. Asci
(the second in the water); N. Ascospores in 2% KOH reagent. Scale bars: 1 mm (C); 700 um (D); 1 mm (E); 30 um (F-H);

15 um (1); 40 um (J); 50 um (K); 150 um (L, M); 100 um (N).

MycoKeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642 282

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

Description. Saprobic on the bark of the living Fagaceae tree. Sexual morph:
Apothecia 1.9-3.2 mm wide (x = 2.6 um, n = 30) when fresh, 1.6-2.6(-3.1)
mm wide (x = 2.1 um, n = 20), (0.5-)0.7-1.0 um high (x = 1.6 um, n = 20)
when dried, scattered, superficial, cupulate, stipitate, erumpent from the bark,
initially growing as a cleistohymenial development, obconical without a point,
the hymenium tightly protected by excipulum when immature, splitting to ex-
pose hymenium by eight teeth-like lobes in the surface in a humid environment,
black (#4a4750) surface with polygonal areolae, becoming warty bulges after
opening. Discs flat to slightly detained in the centre, circular to irregular-shaped,
greyish-white (#abb3b6) when fresh, triangular to angular-shaped, the edges
curling towards the centre, desaturated dark green (#9eb07c) when dried. Re-
ceptacles rough and black when fresh, sculptured with polygonal areolae when
dried. Stipes 0.5-1.0 mm wide, 0.6-1.0 mm high when dried, concolorous to
the receptacles. Lips absent. Covering stroma 63-138 um thick, comprised of
carbonised, textura angularis cells and the inner layers hyaline, textura angu-
laris to globulosa cells. Hymenium 378-432 um (x = 399 um, n = 20) thick,
hyaline. Subhymenium 52-111 um (x = 85 um, n = 40) thick, comprised of
hyaline, textura globulosa to angularis cells, 4.5-9.5(-—11.3) um (x = 7.1 um, n
= 80) in diam. Internal matrix of stroma 135-220 um (x = 180 um, n = 40) thick,
well-developed, non-gelatinous, divided into three parts, part | near subhymeni-
um comprised of dense and pale brown, textura intricata hyphae, 1.5-2.5 um
(x = 2.0 um, n = 40) in diam.; part Il in the middle, 37-74 um wide, comprised
of carbonised, black red (#28171a), textura angularis cells same as the ectal
excipulum; part III (stipe) comprised of hyaline and densely parallel hyphae,
mixed with large refraction resin materials, hyphae 1.7-2.5 um (x = 2.1 um,
n = 60) in diam. Basal stroma 65-140 um (x = 101 um, n = 60) thick, well-de-
veloped, the outer layers comprised of carbonised, black red, textura angularis
cells, 2.9-5.8 um (x = 4.3 um, n = 100) in diam., the inner layers comprised
of hyaline cells, 5.5-10.3(-12.7) um (x = 8.0 um, n = 80) in diam., partial ele-
ments orientated at a high angle or vertical to receptacle surface, slightly ge-
latinous. Paraphyses 315-345 x 1.7-2.7 um (x = 332 x 2.1 um, n = 40) wide,
hyaline, filiform, unbranched, aseptate, apically rounded, slightly swollen and
waved, surrounded by a thin, gelatinous sheath. Asci ripening sequentially,
272-355(-373) x 21-30 um (x = 312 x 25 um, n = 40), unitunicate, 8-spored,
clavate, apically ellipse, without amyloid reaction in Melzer’s reagent, tapering
to a fragile pleurorhynchous, subtruncated base, croziers absent. Ascospores
187-226(-241) x 6.8-10.5 um (x = 206 x 7.9 um, n = 40, Q = (17.8)22.9-31.3,
Qm = 26.3 + 3.1), overlapping fascicles, long acicular, transverse-septate, hya-
line, 24-33(-—38)-septate when mature with a single oil drop in each cell, sharp
ends, wide at the top and tapering downwards, thin and rough-walled with fine
verrucae. Asexual morph: Undetermined.

Material examined. CHINA * Yunnan Province, Dali City, Jinguangsi Protec-
tion Zone, altitude 2,380 m, on the living bark of Fagaceae tree, 27 July 2024,
Cuijinyi Li LCUY-1695 (holotype HKAS 145641); « ibid., 24 July 2024, Cuijinyi Li
LCJY-1642 (paratype HKAS 145639).

Notes. Triblidium stipitatum is distinguished from all other known species by
its well-developed stipe and the special excipulum structure. It is closely relat-
ed to T. longisporum, based on both morphological and phylogenetic analysis,
but can still be easily distinguished. Triblidium stipitatum can be differentiated

MycoKeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642 283

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

from the latter species by the presence of stipes, greyish-white hymenium, a
basal stroma consisting of three parts (from top to bottom: pale brown intri-
cate hyphae, black red angularis cells and hyaline parallel hyphae mixed with
large refraction resin materials), as well as slightly longer asci (272-355 um vs.
250-336 um) and ascospores (187-226 um vs. 160-196 um). The molecular
analyses were shown in the note of T. longisporum.

Discussion

Previous studies emphasised the use of variations in ascospore morphology
for differentiating species within Triblidium (Eriksson 1992; Karakehian et al.
2019); however, this approach has often led to overestimations of taxonomic
diversity and frequent revisions in classification (Lantz et al. 2011; Karakehian
et al. 2019). The reliance on ascospore characteristics alone has proven insuf-
ficient for stable and accurate taxonomic delineation, highlighting the need for
a more comprehensive evaluation of additional morphological and molecular
features. Based on the current phylogenetic analysis, the elongated and slightly
curved fusiform ascospores of H. verrucosa (now recognised as T. verruco-
sa) are distinct from the ellipsoid and muriform ascospores observed in oth-
er species, including the type species (Karakehian et al. 2019). Nevertheless,
T. verrucosa is regarded as a member of Triblidium to maintain the monophy-
letic status of the genus. After incorporating two newly-described species with
long acicular ascospores into the phylogenetic analysis, T. verrucosa continues
to cluster within Triblidium. Similarly, T. stipitatum, T. longisporum and T. calici-
iforme (the type species) cluster in the basal or lower clades. Despite its diver-
gent morphology, T. verrucosa was still retained within Triblidium to maintain
the monophyletic integrity of the genus (Lv et al. 2019).

We propose potential directions for future taxonomic studies here. Guo et
al. (2024) suggested that Triblidium is a potential plant-associated ascomyce-
te, with seven species reported from Rhododendron sp. (Ericaceae) and oth-
ers isolated from branches of Fagaceae, Pinaceae and Salicaceae. As shown
in Fig. 1, the phylogenetic tree reveals the following groups: Group | includes
one species from Fagaceae, characterised by orange hymenium and ellipsoid,
muriform ascospores; Group II comprises six species from Rhododendron
sp., also with orange hymenium and ellipsoid, muriform ascospores; Group III
contains a single species from Pinaceae, distinguished by orange hymenium,
elongated and slightly curved ascospores with transverse septa, fine verrucae
on the surface and distinctive round appendages at both ends; Group IV en-
compasses two species from Fagaceae, with greyish-white or pale orange hy-
menium and long acicular ascospores that exhibit transverse septa and fine
verrucae; Group V includes one species from both Fagaceae and Salicaceae,
notable for black hymenium and ellipsoid, muriform ascospores; Group VI rep-
resents an unidentified species from Fagaceae, exhibiting translucent yellow
hymenium and ellipsoid, transversely stipitate ascospores. Although the asco-
spores in Groups III and IV are morphologically unique within Triblidium, they
are phylogenetically closely related to species with ellipsoid ascospores in our
study. Other micro-morphological characteristics did not facilitate delineating
the clades. Thus, integrating plant associations, ascospore morphology and
phylogenetic analysis supports these clades. The limited number of available

MycoKeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642 284

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

species constrain comprehensive phylogenetic analyses. This study relied
solely on ITS, LSU and mtSSU regions and the current database lacks infor-
mation from additional genomic regions, such as protein-coding genes (e.g.
rpb2, tef1), which are essential for comprehensive phylogenetic analyses and
might lead to the further confirmation of the monophyly of Triblidium or po-
tential division. Future research incorporating more diverse morphological and
molecular data will likely yield more understanding of the interspecific relation-
ships within Triblidium.

At the same time, it remains debatable whether excessive emphasis has
been placed on the study of spore morphology. From a broader view, all mem-
bers of the family share carbonised black ascomatal walls, which serve to
protect the delicate spore layer in arid environments (Karakehian et al. 2019).
However, variations in spore morphology may reflect diverse survival strate-
gies. For example, muriform ascospores may exhibit greater adaptability in
harsh terrestrial ecosystems; the large spores tend to be deposited over short
distances and are less sensitive to desiccation and UV radiation, while small
spores are more suited for long-distance dispersal via air or water. Thick-walled
spores offer resistance to dry environments and the surface ornamentation
can reduce settling velocities and influence trophic method (Karakehian et al.
2019; Quijada et al. 2022). Spore morphology could offer insights, potentially
prompting a re-evaluation of current taxonomic classifications. This may re-
sult in the re-organisation of existing taxa and the establishment of additional
genera within Triblidiaceae.

Acknowledgements

Cui-Jin-Yi Li would like to thank Mae Fah Luang University for granting Ph.D.
studies. K.D. Hyde was funded by the Distinguished Scientist Fellowship Pro-
gram (DSFP), King Saud University, Kingdom of Saudi Arabia and thanks the
Chinese Research Fund (project no E1644111K1) entitled “Flexible introduc-
tion of the high-level expert program”, Kunming Institute of Botany, Chinese
Academy of Sciences.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Use of Al

No use of Al was reported.

Funding

This study is supported by the Major Science and Technology Special Project of Yunnan
Province (202502AE090044) and the Innovation Guidance and Technology-Based
Enterprise Cultivation Program of Yunnan Province Science and Technology Department
(No. 202504BI090008).

MycoKeys 121: 271-289 (2025), DOI: 10.3897/mycokeys.121.165642 285

Cui-Jin-Yi Li et al.: Three new taxa of Triblidium from west Yunnan, China

Author contributions

All authors have contributed equally.

Author ORCIDs

Cui-Jin-Yi Li © https://orcid.org/0000-0002-2805-7071

Qi Zhao ® https://orcid.org/0000-0001-8169-0573

Prapassorn Damrongkool Eungwanichayapant © https://orcid.org/0000-0001-8005-4137
Feng-Ming Yu ® https://orcid.org/0000-0001-9133-8645

Kevin David Hyde © https://orcid.org/0000-0002-2191-0762

Kandawatte Wedaralalage Thilini Chethana © https://orcid.org/0000-0002-5816-9269
Wei-Wei Liu © https://orcid.org/0000-0001-5670-606X

Dong-Mei Liu © https://orcid.org/0000-0001-9190-8871

Data availability

All of the data that support the findings of this study are available in the main text.

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